Derivatives of n(n-acyl amino acyl) aminoacetonitrile

ABSTRACT

N-(N-acylaminoacyl)-aminoacetonitriles of the formula   D R A W I N G

United States Patent Irikura et al. 1 Oct. 10, 1972 [s41 DERIVATIVES 0F N(N-ACYL AMINO 3,211,779 10/1965 Nowak ..260/465.4 ACYL) AMINOACETONITRILE 3,511,867 5/1970 Laliberte ..260/465.4

[72] Inventors: Tsutomu Irikura; Keigo Nishino; Seigo Suzue; Keichi Ushiyama, all of Tokyo; I-Iirotaka Shinada, Saitamaken, all of Japan [73] Assignee: Kyorin Seiyaku Kabushiki Kaisha,

Tokyo, Japan [22] Filed: May 28, 1969 [21] Appl. No.: 828,508

Related U.S. Application Data [63] Continuation-in-part Ser. No. 449,472, Apr.

20, 1965, Pat. No. 3,467,691.

[30] Foreign Application Priority Data April 22, 1964 Japan ..39/22575 [52] U.S. Cl ..260/465.4 [51] Int. Cl. ..C07c 121/44, C070 103/30 [58] Field of Search ..260/404.5, 465.4; 424/304 [56] References Cited UNITED STATES PATENTS 2,461,842 2/ 1949 Olin ..260/465.4 3,201,458 8/1965 Scheurer et al ..260/465.4 3,206,491 9/1965 Gubler et a1 ..260/465.4

3,000 I 1.0 I 1 2 I 1 N I Z I lLl I 2,000 m l z D I:

- .l l .L I L I N-(N-acylaminoacyl)-aminoacetonitri1es of the formula 7 R CO--NHCHz-CN NH-CO-R m which R-CO NH- 15 the radlcal of R-OOOH representing an amino acid selected from the group consisting of glycine,a-alanine, B-alanine, aaminoisobutyric acid, valine, methionine, lysine, aspartic acid, and S-aminovaleric acid and R CO represents an acyl radical, R representing a member selected from the group consisting of straight and branched chain alkyl radicals having from one to four carbon atoms.

15 Claims, 2 Drawing Figures TENDENCY OF GLUTAMIC OXALOACETIC TRANSAMINASE IN BLOOD SERUM -----HYPODERMIC ADMINISTRATION OF 100 mg/Kg CARBON TETRACHLORIDE (mouse) SIMULTANEOUS ADMINISTRATION OF 100 mg/kg ACETYL GLYCINYL AMINOACETONITRILE AND 100 mg/kg CARBON TETRACHLORIDE (mouse) UNITS OF ENZYME UNITS 0F ENZYME PATEIITEDIIBI 10 1972 TENDENCY 0F GLUTAMIC OXALOACETIC. TRANSAMINASE IN BLOOD SERUM '---.HYPODERMIC ADMINISTRATION OF I00 mg/kg CARBON TETRACHLORIDE (mouse) HSI MULTAN EOUS .ADMINIS'I'RATION OF'IOO rng/kg ACETYL GLYCINYL AMINOACETONITRILE AND I00 mg/kg CARBON TETRACHLORIDE (meuse) TENDENCY 0F GLUTAMIC PYRUVIC TRANSAMINASE IN BLOOD SERUM -----ADMINISTRATION OF I00 mg/kg CARBON TETRACHLORIDE (mouse) Tsqfcmu lrjkura, K0190 N/shmo, Selgo Suzue,

Kel'chi Ush/yama ana Hirofaka Shinodo INVENTORS 1 2 DERIVATIVES F N(N-ACYL AMINO ACYL) Liver Przslappressing kathyrogenic tectin e ect on ctivity P T l l as. m... This application 18 a contmuatton-m-part of applica- Activity Yerrnicula- M tion Ser. No. 449,472, filed Apr. 20, 1965 now US. Pat. No. 3,467,691. 5 The present invention relates to derivatives of N-(N- g noaceao it i e d IS ton 33 Very Strong mino aci acy ate qua to 0 Half of acylammoacyl) aminoacetonitriles of the l Aminoacetonitrile Aminoacetoni- Aminoaceto- Aminoacetotrile nitrile nitrile f C0 NH CH2 ON Acyl Aminoacid Egu altg Amino- Less than Less than 1 Acylated Aminoacetonitrile l/ 10 of A of Aminoin which acetonitrile of Aminoacetoacetonitrile R 0 Present Invention nitric NH- is the residue of R-COOH It should be noted that liver cirrhosis, namely fibrination cannot be suppressed by the complete removal of lathyrogenic activity. Thus, where giving priority to the liver function (for example in cases where it is desired to prevent liver disorder caused by inspirated anesthesia, blood transfusion and so on), a lower lathyrogenic activity is advantageous. This means that the substances of the present invention are more useful in this respect. Meanwhile, where preventing liver cirrhosis tendencies, amino acid acylated aminoacetonitrile is more advantageous.

The preventive effect on liver disorder caused by carbon tetrachloride, of acetyl glycinyl aminoacetonitrile, the simplest one of the substances of the present invention, will be described by way of ex- 0 ample, referring to the accompanying drawings.

FIG. 1 and FIG. 2 show the preventive effect, on liver disorder caused by carbon tetrachloride, of a representative substance of the present invention, i.e., acetyl glycinyl aminoacetonitrile.

The two kinds of transaminase shown in FIG. 1 and FIG. 2 will be released in a large amount into the blood serum when the parenchyma of the liver is broken. On the other hand it has been proved by various experiments that the absence of these enzymes in the blood serum indicates no disorder in the parenchyma of the liver. Thus, this has been adopted as a most reliable method for the diagnosis of human liver disease.

representing amino acids such as glycine, a-alanine, B-aIanine, a-aminoisobutyric acid valine, methionine, lysine, tryptophane 13-, aspartic acid, phenylalanine, tyrosine, phenyl glycine, 8-amino-valeric acid, etc., and R'-CO represents an acyl radical, R representing a straight or branched chain alkyl radical or an aromatic radical.

The present invention is based on our discovery that the secondary effects of an amino acid acylated aminoacetonitrile can be reduced without lowering its liver protecting effects by acylation of the amino radical thereof.

Some of the secondary effects of amino acid acylated aminoacetonitriles will be described in more detail 3 hereinafter.

Aminoacetonitrile, which is the starting substance for the preparation of amino acid acylated aminoacetonitrile, not only has such strong medical effects that it completely prevents liver damage caused by liver damaging substances such as carbon tetrachloride, bromobenzene, thioacetoamide, etc., but it also is capable of regenerating the liver and of preventing liver cirrhosis. On the other hand, however, it has harmful pharmacodynamical effects on the vermiculation of intestines and a strong influence both on the heart rate and the blood pressure to the extent that 7 this substance has no value as medicine. It can bc Seen from 1 and 2 that acetyl The aforementioned amino acid acylated glycinyl aminoacetonitrile completely prevents the aminoacetonitrile has the same degree of liver protect- 5 hvel' dlsorder whlch; Otherwlse caused y 100 mg/kg ing effect as aminoacetonitrile; and it has become ofcarboh tetrachlohda possible to reduce its pharmacodynamical virulence to The substances to which the P invention less than one-tenth. Thus, it has become useful as ates are 'syhthesized y y g amino acid acylated aminoacetonitrile with a suitable acylating medicine.

However some interesting facts have been found 2 such as, for pl ahhydhde acld hahde, from biochemical studies on these substances. f ester Namely, it has been found that the so-called y be shown by fl as follows! lathyrogenic substance present in the sweet pea causes OHN CHZ CN O denaturation (hquescence) of acid muco polysaccharide which is a component of young animals cartilage, thuscausmg denaturatlon of the cartilage PFCONHCHPON and, in addition, suppressing the propagation of fibrinorous cells. It is also known that in which aminoacetonitrile is one of the strongest substances 6O R known with respect to lathrogenic activity.

The present inventors have conducted studies for is the residue of overcoming the correlation between liver protection activity, pharmacodynamic activity, and lathyrogenic NH:

activity, and have now succeeded in breaking off the 65 representing an amino acid correlation and applying only the useful activities 7 thereof to the medical treatment of human diseases. R represents a straight or branched chain alkyl, or an The above developments are tabulated as follows: aromatic radical and X represents halogen.

1 1112 0 (or moo-x R-COOH hydride,

When an acid anhydride is used as the acylating agent, the amino radical is easily acylated by reaction of an amino acid acylated aminoacetonitrile (or its acetate) with an acid anhydride such as acetic anpropionic anhydride, butyric anhydride, isobutyric anhydride, etc., without a solvent or in a suitable solvent (preferably aqueous solution), so that the desired acyl amino acid acylated aminoacetonitrile, N-(N-acetyl glycyl)-aminoacetonitrile, for example, is

obtained.

When an acid halide, such as acetyl chloride, acetyl bromide, propionyl chloride, butyryl chloride, etc., is used "as the 1acylating agent, it is desirable that a neutralizing agent; such as sodium bicarbonate, sodium carbonate, potassium carbonate, pyridine, triethylamine, etc., be concomitantly used, and that the reaction be carried out in an aqueous solution or in an organic solvent (ether, benzene, alcohol, pyridine, for example). It isparticularly advantageous in this case that the reaction take place in an aqueous solution at a temperature between 25 C.

The present invention is hereinafter described in more detail setting forth presently preferred examples.

Amino acid acylated acetonitrile (II) or a derivative thereof, which is the starting material for the present invention, is produced by a method, for example, in which an acid amide synthesized from phthaloyl amino acid and aminoacetonitrile, having the general formula (III), is subjected to the action of hydrazine or its hydrate:

(III) l NH2-NH2-H2 acid, such as its halide, with aminoacetonitrile. This is shown by the following reaction scheme:

NRG OHal-HzNCIhCN c I O i II o N-RCONHCH2CN o EXAMPLE 1 N-( N-Acetyl Glycyl)-Aminoacetonitrile One g (gram) of N-glycylaminoacetonitrile (or its acetate) is dissolved in 5 cc (cubic centimeters) of water, and 1.5 cc of acetic anhydride is added at one time at room temperature (20 to 30 C) with stirring. After 15 minutes, 1.5 cc of acetic anhydride is again added with stirring for 30 minutes thereafter, and then condensation under diminished pressure is effected. When the resultant crystals are subjected to recrystallization from ethanol 0.8 g of fine plate crystals are obtained. The melting point of this substance is l57l 5 9 C.

Analysis: N: I

Glycyl-aminoacetonitrile used as starting material in this example can be produced as follows:

Five g of aminoacetonitrile sulphate is dissolved in 100 cc of water, with addition of 12 g of sodium bicarbonate. The resulting solution is cooled to 5 C and 50 cc of dioxan solution of 10 g of phthaloyl glycyl chloride is dropped thereintowithstirring for a period of 30 minutes, during which period crystals precipitate. Then stirring is continued at 5 C for 2 hours and crystals are collected by filtration,- washed with water and dried.

White needle crystals are obtained .by recrystallization from acetonitrile. The melting point of this substance is between 245 C and 246 C. Analysis: C' H bl3N Calculated: N 17.28 Found: 17.57.

Then 7 g of phthaloyl glycyl aminoacetonitrile is added to 50 cc of ethyl alcohol solution of 18 g of 80 percent hydrazine hydrate and heated for 1.5 hours to distil off ethyl alcohol under diminished pressure. The residue is added to cc of aqueous solution of g of cone. hydrochloric acid and the solution is stirred for 5 minutes and cooled. Then undissolved crystals are removed and when the solution is condensed under 40 C, a syruplike residue is obtained.

The residue is dissolved in theleast possible amount of methanol .with addition of ethyl alcohol. When this solution is left in a refrigerator, 3 g of white crystal of the object substance is obtained.-

The melting point of the substance is between l79l83 C. Analysis: C H N Cl Calculated: N 28.05 Found: N 28.87.

EXAMPLE 2 N-(N-Acetyl Glycyl)-Aminoacetonitrile One g of glycyl-aminoacetonitrile acetate is dissolved in 5 cc of water. Two g of sodium bicarbonate is added and then 1 cc of ether solution of 0.5 g of acetylchloride is dropped into the solution at a temperature between 0 and 5 C while stirring and cooling the solution with ice water. Stirring is continued at a temperature between 0 and 5 C for 30 minutes thereafter, and further stirring is continued at room temperature for 1 hour. Then the reaction solution is filtered and when the filtrate is subjected to condensation under diminished pressure below 40 C, there are left crystals.

These crystals are extracted with hot ethanol, andv undissolved substances are filtered off. When the filtrate is cooled, white plate crystals are obtained. The melting point of this substance is l57l50 C and the amount obtained is 0.6 g. When this substance is mixed with that obtained by Example I, no lowering of the melting point is observed.

EXAMPLE 3 N-(N-Acetyl-Valyl)-Aminoacetonitrile 'yNH 3380, 3020; 1650 6NH 1530, 1550;

y C=O a CH,

In this way the crystals are determined to be the object substance.

EXAMPLE 4 N-( N-Acetyl-a-Alanyl )-Aminoacetonitrile One-hundred mg of N-a-alanyl aminoacetonitrile acetate and 0.15 cc of acetic anhydride are caused to react and treated in a similar way as shown in Example 1 to obtain the object substance. Recrystallized from isopropyl alcohol, 60 mg of white needle crystals, having a melting point between l49-150 C, are obtained.

lnfrared Analysis (cm) NH 3380 3300 C=Q 1680 1640 NH 1570 1540 CH 1380 1260 Thus, the crystals are determined to be the object substance.

EXAMPLE 5 White needle crystals of N(N-propionyl-glycyl)- aminoacetonitrile having a melting point between 150 and 160 C are obtained similarly, using ethanol as a recrystallization solvent.

EXAMPLE 6 White needle crystals of N-(N-n-butyryl-glycyl)- aminoacetonitrile (M.P. l29-l 3 1 C) are similarly obtained, using ethanol.

EXAMPLE 7 White needle crystals of N-(N-acetyl-alanyl)- aminoacetonitrile (M.P. l49-l50 C) are similarly obtained, using isopropyl alcohol.

EXAMPLE 8 White needle crystals of N-(N-isobutyryl-alanyl)- aminoacetonitrile (M.P. l65-l66 C) are similarly ob tained, using cthunol.

EXAMPLE 9 White needle crystals of N-(N-propionyl-valyD- aminoacetonitrile (M.P. l8 -l 85 C) are similarly obtained, using ethanol.

EXAMPLE 10 White needle crystals of N-(N-butryl-valyl)- aminoacetonitrile (M.P. 172-l 73 C) are similarly obtained, using 30 percent ethanol.

EXAMPLE 1 l White needle crystals of N-(N-isobutyryl-valyl)- aminoacetonitrile (M.P. l92-193 C) are similarly obtained, usingethanol h M EXAMPLE 12 White needle crystals of N-(N-acetyl-B-alanyD- aminoacetonitrile (M.P. l43-l44 C) are similarly obtained, using ethanol.

EXAMPLE 13 White needle crystals of N-(N-acetyl-methionyl)- aminoacetonitrile (M.P. l09-l 1 1 C) are similarly obtained, using isopropyl-alcohol.

EXAMPLE 14 White needle crystals of N-(N-acetyl-asparaginyl)- aminoacetonitrile (M.P. l 96 C) are similarly obtained, using water-ethanol.

EXAMPLE 15 White leaflet crystals of N-(N-acetyl-8- aminovaleryl)-aminoacetonitrile (M.P. 101 C) are similarly obtained, using ethyl acetate.

The safety and effectiveness of the N-(N- acylaminoacyl)-aminoacetonitriles of the present invention as medicaments will be apparent from the following data.

In Table l the effect of the compositions of the present invention on the Lathyrism and body weight in rats as compared with conventional compositions is set forth. The table also demonstrates the effect of the compared compositions on the mortality of the rats.

TABLE 1 Determination of Lathyrogenic Activities (young rats, ten-day administration) Body Weight Determination of Mortality lncrease/ Lathyrism by day X-ray" Normal rat 0 5.3 Aminoacetonitrile [00 l .4 l+ls/ s) Glycyl Aminoacetonitrile 10 -0.2 -H- (200mg/Kg) Glycyl Aminoacetonitrile I00 O.2 -H-+ (SOOmg/Kg) Acetyl Glycyl Aminoacetonitrile 0 3.9

M/ s) Benzoyl Glycyl Aminoacetonitrile 0 5.2

(l s/ s) '-not lathyritic; clearly lathyritic; 4-H- vcry severely lathyritic;

slightly lathyritic; ++severely lathyritic The serum-glutamic-oxaloacetic trans-aminase (hereinafter abbreviated as S-GOT) activity and serum-glutamic-pyruvic transaminase (hereinafter abbreviated as S-GPT) activity suppressing effect of the compounds of the invention were determined in the following manner.

Mice were hypodermically injected with an amount corresponding to 0.1 ml/kg of carbon tetrachloride dissolved in olive oil and an amount corresponding to 100 mg/kg of the compounds of the invention, the difficultly soluble compounds being orally dosed. A control group of mice were injected only with carbon tetrachloride. The S-GOT and S-GPT elevation rates of the control group were appraised at 100 percent each.

The corresponding values for each group dosed with the compoundsof the invention are also set forth in terms of their elevation rate. In addition, the elevation rates of a normal group of mice which were not injected with carbon tetrachloride are also set forth.

The results are set forth in Table 2. In table 3, the results achieved by varying the dosages of the compounds of the invention are set forth.

The data in the Tables below are indicative of the effect of some of the compounds according to the present invention on carbon tetrachloride liver disordered mice.

TABLE 2 S-GOT S-GPT Acute poison to elevation elevation mouse, venous rate rate injection, LD

Group 5 I: mg/kg Control Group 100 100 50 Normal group 3.62.0.7 0910.8 N-Acetylglycylamino acetonitrile 1 1.8:26 2.6109 4000 N-Acetylvalylamio acetonitrile 6.5122 1511.4 N-Acetylmethionylacetonitrile 266:2.8 4.6:05 N-Acetylvalylamio caproylaminoacetonitrile 27.3:3 .0 28.4fl.8 3000 N-Propionylglycylaminoacetonitrile l 3.4:] .9 l .1104 N-n- Butyrylglycylamino acetonitrile l4.6.t3.5 3,211.3 N-Acrylylglycylaminoacetonitrile 3.0:11 16.7106 N-Benwylglycylaminoacetonitrile 3.9108 1.6106

TABLE 3 N-lsobutyrylalanyl aminoacetonitrile. Serum-Gotk Serum GPT normal 32.2 1.02 C C14 100 100 50mg/kg+C C14 47.7 2.04 lOOmgIkg-l-C C14 37.6 1.02 200mg/kg-1-C C14 .1 43.5 1.02

N-acetyl phenyl al-.

SOrng/kg-tC C14 62.4 61.8 IOOmgIkg+C C14 29.8 26.9 ZOOmg/kg-t-C C14 27.0 32.5 400mgIkg+C C 14 21.3 3.4 N-acetyl methionyl AAN (S.C.) Serum-GOT I: Serum-OPT normal 3.6 0.9 C C14 100 IOOmg/kg-l-C C14 26.6 4.6 N-ncetyl asparagyl AAN (S.C.) Serum-GOT Q Serum-OPT '5 normal 3.6 0.9 C C 14 100 100 IOOmgIkg-l-C C14 15.3 8.7

CC14 0.1ml/kg. 17 24 hr GOT: Glutamic oxalacetic transaminue GPT: Glutamic pyruvie transaminue What is claimed is: l. N-(N-acylaminoacyl)-aminoacetonitrile of the formula RCO-NH"CHCN NH-COR in which R-GO 7 NH- is the radical of R-COOH representing an amino acid selected from the group consisting of glycine, a-alanine, fl-alanine, aaminoisobutyric acid, valine, methionine, lysine, aspartic acid, and S-aminovaleric acid and R'CO represents an acyl radical, R representing a member selected from the group consisting of straight and branched chain alkyl radicals having from one to four carbon atoms.

2. A compound as in claim 1, said compound being N-(N-acetyl-glycyl)-aminoacetonitrile.

3. A compound as in claim 1, said compound being N-(N-acetyl-valyl)-aminoacetonitrile.

4. A'compound as in claim 1, said compound being N-(N-acetyl-a-alanyl)-aminoacetonitrile.

5. A compound as in claim 1, said compound being N-(N-propionyl-glycyl)-aminoacetonitrile.

6. A compound as in claim 1, said compound being N-(N-n-butyryl-glycyl)-aminoacetonitrile.

7. A compound as in claim 1, said compound being N-(N-acetyl-alanyl)-aminoacetonitrile.

8. A compound as in claim 1, said compound being N-(N-isobutyryl-alanyl)-aminoacetonitrile.

9. A compound as in claim 1, said compound being N-(N-propionyl-valyl)-aminoacetonitrile.

10. A compound as in claim 1, said compound being N-(N-butyryl-valyl)-aminoacetonitrile.

11. A compound as in claim 1, said compound being N-(N-isobutyryl-valyl)-aminoacetonitrile.

12. A compound as in claim 1, said compound being N-(N-acetyl-B-alanyl)-aminoacetonitrile.

13. A compound as in claim 1, said compound being N-(N-acetyl-methionyl)-aminoacetonitrile.

14. A compound as in claim 1, said compound being N-(N-acetyl-asparaginyl)-aminoacetonitrile.

15. A compound as in claim 1, said compound being N-(N-acetyl-S-aminovaleryl)-aminoaceton itrile. 

2. A compound as in claim 1, said compound being N-(N-acetyl-glycyl)-aminoacetonitrile.
 3. A compound as in claim 1, said compound being N-(N-acetyl-valyl)-aminoacetonitrile.
 4. A compound as in claim 1, said compound being N-(N-acetyl-a-alanyl)-aminoacetonitrile.
 5. A compound as in claim 1, said compound being N-(N-propionyl-glycyl)-aminoacetonitrile.
 6. A compound as in claim 1, said compound being N-(N-n-butyryl-glycyl)-aminoacetonitrile.
 7. A compound as in claim 1, said compound being N-(N-acetyl-alanyl)-aminoacetonitrile.
 8. A compound as in claim 1, said compound being N-(N-isobutyryl-alanyl)-aminoacetonitrile.
 9. A compound as in claim 1, said compound being N-(N-propionyl-valyl)-aminoacetonitrile.
 10. A compound as in claim 1, said compound being N-(N-butyryl-valyl)-aminoacetonitrile.
 11. A compound as in claim 1, said compound being N-(N-isobutyryl-valyl)-aminoacetonitrile.
 12. A compound as in claim 1, said compound being N-(N-acetyl-Beta -alanyl)-aminoacetonitrile.
 13. A compound as in claim 1, said compound being N-(N-acetyl-methionyl)-aminoacetonitrile.
 14. A compound as in claim 1, said compound being N-(N-acetyl-asparaginyl)-aminoacetonitrile.
 15. A compound as in claim 1, said compound being N-(N-acetyl-delta -aminovaleryl)-aminoacetonitrile. 